A biosensor is an analytical device that incorporates a biological recognition element in direct spatial contact with a transduction element. That integration ensures the rapid and convenient conversion of biological events to detectable signals. Among diverse electrical biosensing architectures, devices based on field-effect transistors (FETs) have attracted great attention because they are a type of biosensor that can directly translate interactions between target molecules (e.g., biological molecules) and the transistor surface into readable electrical signals. In a standard field effect transistor, current flows along a conducting path (the channel) that is connected to two electrodes, (the source and the drain). The channel conductance between the source and the drain is switched on and off by a third (gate) electrode that is capacitively coupled through a thin dielectric layer. Field-effect transistors detect target chemicals and measure chemical concentrations for a wide range of commercial applications including, for example, industrial process control, leak detection, effluent monitoring, and medical diagnostics.
A problem with field-effect transistors is their limit of sensitivity. Field-effect transistors are not able to accomplish single molecule detection, i.e., these transistors are not able to detect at the level of one single molecule. Additionally, these transistors are not able to monitor the dynamics of a single molecule reaction. The sensitivity limitation of field-effect transistors prevents their use as detectors in important biochemical assays, such as detectors in a single molecule sequencing reaction.
Improving upon sensitivity has been explored in the past using devices based on single-walled carbon nanotubes (SWNTs). See A. Star et al., Nano. Lett. 3, 459 (2003); A. Star et al., Org. Lett. 6, 2089 (2004); K. Besterman et al., Nano. Lett. 3, 727 (2003); G. Gruner, Anal. Biooanal. Chem. 384, 322 (2005); R. Chen et al. Proc. Natl. Acad. Sci. U.S.A. 100, 4984 (2003). The motivation for using SWNTs in sensor FETs is that SWNTs are extremely small conductors, typically only 1 nanometer in diameter.
Past research has coated SWNT sensor FETs with chemoselective polymers, metal and metal oxide nanoparticles, and biomolecules like proteins and antibodies. These sensitizing molecules or sensitizing agents direct the innate sensitivity of the SWNT towards a particular chemical target. Past work has exclusively used coatings of sensitizing agents in which numerous molecules were attached to the SWNT. See K. Besterman et al., Nano. Lett. 3, 727 (2003); R. Chen et al. Proc. Natl. Acad. Sci. U.S.A. 100, 4984 (2003). Prior work, however, has lacked any method to controllably attach single sensitizing molecules to the SWNT, and the use of multiple sensitizing molecules typically resulted in a mixture of true signal and ensemble properties. This has complicated the analysis of any data acquired from the sensor FET, and precluded the application of probing a single molecule's dynamics.
The state of the art in improving this embodiment to single molecule sensitivity has used a special technique for creating one single covalent defect on the SWNT. See Goldsmith et al. Science 315, 77 (2007). Once the SWNT contains a single defect, a variety of attachment chemistries can be chosen which link to the reactive defect site selectively, without coating the rest of the SWNT with additional sensitizing molecules. This method of fabrication previously relied on electrochemical oxidation of the SWNT, creating a defect site on the wall of the SWNT constituting a functional group, followed by covalent functionalization of the sensitizing molecule to the defect site functional group. See Goldsmith et al, Nano Letters 8, 189 (2008); Coroneus et al. ChemPhysChem 9, 1053 (2008); Sorgenfrei et al., Nat. Nano. 6, 126 (2011). This method provides a single molecule device that is sensitive to dynamic fluctuations. The SWNT defect also invariably results in a drop in the conductivity of the SWNT and an increase in device noise, both as a result of the necessary disruption of the SWNT's sp2 conjugation and aromaticity. Reports of this technique indicate that when electrochemical oxidation is terminated to result in a 90% reduction in conductivity, 88% of the devices remain conductive, but of those only 19% of devices yield functional devices with single sensitizing molecules attached. When electrochemical oxidation is terminated at greater than a 99% reduction in conductivity, only 18% of the devices remain conductive, and of those only 28% yield functional single sensitizing molecule devices. Coroneus et al. established process controls that achieved higher yields approaching 40%. Nevertheless, devices fabricated using this method usually display great chemical variability near the defect site, including broken carbon-carbon bonds which may be tautomerized or protonated, creating high variability among the electronic characteristics of different devices. Furthermore, devices of this type can only be fabricated serially, one device at a time. No methods exist for producing multiple, single-molecule devices in parallel.
SWNTs can also be tailored with sensitizing molecules by non-covalent means. In a non-covalent scheme, sensitizing molecules are weakly bound to SWNTs, thus preserving the sp2 SWNT structure and resulting in more consistent electronic characteristics. See Chen et al, J. Am. Chem. Soc. 123, 3838 (2001). However, such methods do not reveal a method to reliably bind a single sensitizing molecule non-covalently to a SWNT, nor does the prior art demonstrate any device that utilizes a single, non-covalently bound sensitizing molecule.
Consequently, there remains a need for electronic devices that can achieve single molecule dynamic sensing, especially if those devices can be fabricated in greater yields, with chemical functionality, and with more consistent electronic characteristics. Potential applications of a robust system which is capable of the long-term probing and detecting of the dynamics of single molecules could include environmental detection, medical diagnostic tools, biomolecule sequencing such as DNA or RNA sequencing, and other fields of interest, such as security or defense.